The extracellular matrix differentially directs myoblast motility and differentiation in distinct forms of muscular dystrophy: Dystrophic matrices alter myoblast motility.

Extracellular matrix (ECM) pathologic remodeling underlies many disorders, including muscular dystrophy. Tissue decellularization removes cellular components while leaving behind ECM components. We generated "on-slide" decellularized tissue slices from genetically distinct dystrophic mouse models. The ECM of dystrophin- and sarcoglycan-deficient muscles had marked thrombospondin 4 deposition, while dysferlin-deficient muscle had excess decorin. Annexins A2 and A6 were present on all dystrophic decellularized ECMs, but annexin matrix deposition was excessive in dysferlin-deficient muscular dystrophy. Muscle-directed viral expression of annexin A6 resulted in annexin A6 in the ECM. C2C12 myoblasts seeded onto decellularized matrices displayed differential myoblast mobility and fusion. Dystrophin-deficient decellularized matrices inhibited myoblast mobility, while dysferlin-deficient decellularized matrices enhanced myoblast movement and differentiation. Myoblasts treated with recombinant annexin A6 increased mobility and fusion like that seen on dysferlin-deficient decellularized matrix and demonstrated upregulation of ECM and muscle cell differentiation genes. These findings demonstrate specific fibrotic signatures elicit effects on myoblast activity.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

Genetic background shifts the severity of muscular dystrophy. In mice, the DBA/2J strain confers a more severe muscular dystrophy phenotype, whereas the Murphy's Roth Large (MRL) strain has "super-healing" properties that reduce fibrosis. A comparative analysis of the Sgcg null model of Limb Girdle Muscular Dystrophy in the DBA/2J versus MRL strain showed the MRL background was associated with greater myofiber regeneration and reduced structural degradation of muscle. Transcriptomic profiling of dystrophic muscle in the DBA/2J and MRL strains indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized "myoscaffolds". Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix, and dystrophic myoscaffolds from the MRL background were enriched in myokines. C2C12 myoblasts were seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J matrices. Acellular myoscaffolds from the dystrophic MRL background induced myoblast differentiation and growth compared to dystrophic myoscaffolds from the DBA/2J matrices. These studies establish that the MRL background also generates its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

A conserved annexin A6-mediated membrane repair mechanism in muscle, heart, and nerve.

Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 µM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.

Intermittent glucocorticoid treatment enhances skeletal muscle performance through sexually dimorphic mechanisms.

Glucocorticoid steroids are commonly prescribed for many inflammatory conditions, but chronic daily use produces adverse effects, including muscle wasting and weakness. In contrast, shorter glucocorticoid pulses may improve athletic performance, although the mechanisms remain unclear. Muscle is sexually dimorphic and comparatively little is known about how male and female muscles respond to glucocorticoids. We investigated the impact of once-weekly glucocorticoid exposure on skeletal muscle performance comparing male and female mice. One month of once-weekly glucocorticoid dosing improved muscle specific force in both males and females. Transcriptomic profiling of isolated myofibers identified a striking sexually dimorphic response to weekly glucocorticoids. Male myofibers had increased expression of genes in the IGF1/PI3K pathway and calcium handling, while female myofibers had profound upregulation of lipid metabolism genes. Muscles from weekly prednisone-treated males had improved calcium handling, while comparably treated female muscles had reduced intramuscular triglycerides. Consistent with altered lipid metabolism, weekly prednisone-treated female mice had greater endurance relative to controls. Using chromatin immunoprecipitation, we defined a sexually dimorphic chromatin landscape after weekly prednisone. These results demonstrate that weekly glucocorticoid exposure elicits distinct pathways in males versus females, resulting in enhanced performance.

Anti-latent TGFß binding protein 4 antibody improves muscle function and reduces muscle fibrosis in muscular dystrophy.

Duchenne muscular dystrophy, like other muscular dystrophies, is a progressive disorder hallmarked by muscle degeneration, inflammation, and fibrosis. Latent transforming growth factor ß (TGFß) binding protein 4 (LTBP4) is an extracellular matrix protein found in muscle. LTBP4 sequesters and inhibits a precursor form of TGFß. LTBP4 was originally identified from a genome-wide search for genetic modifiers of muscular dystrophy in mice, where there are two different alleles. The protective form of LTBP4, which contains an insertion of 12 amino acids in the protein's hinge region, was linked to increased sequestration of latent TGFß, enhanced muscle membrane stability, and reduced muscle fibrosis. The deleterious form of LTBP4 protein, lacking 12 amino acids, was more susceptible to proteolysis and promoted release of latent TGF-ß, and together, these data underscored the functional role of LTBP4's hinge. Here, we generated a monoclonal human anti-LTBP4 antibody directed toward LTBP4's hinge region. In vitro, anti-LTBP4 bound LTBP4 protein and reduced LTBP4 proteolytic cleavage. In isolated myofibers, the LTBP4 antibody stabilized the sarcolemma from injury. In vivo, anti-LTBP4 treatment of dystrophic mice protected muscle against force loss induced by eccentric contraction. Anti-LTBP4 treatment also reduced muscle fibrosis and enhanced muscle force production, including in the diaphragm muscle, where respiratory function was improved. Moreover, the anti-LTBP4 in combination with prednisone, a standard of care for Duchenne muscular dystrophy, further enhanced muscle function and protected against injury in mdx mice. These data demonstrate the potential of anti-LTBP4 antibodies to treat muscular dystrophy.

A gene-edited mouse model of limb-girdle muscular dystrophy 2C for testing exon skipping.

Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the γ-sarcoglycan (SGCG) gene. The most common SGCG mutation is a single nucleotide deletion from a stretch of five thymine residues in SGCG exon 6 (521ΔT). This founder mutation disrupts the transcript reading frame, abolishing protein expression. An antisense oligonucleotide exon-skipping method to reframe the human 521ΔT transcript requires skipping four exons to generate a functional, internally truncated protein. In vivo evaluation of this multi-exon skipping, antisense-mediated therapy requires a genetically appropriate mouse model. The human and mouse γ-sarcoglycan genes are highly homologous in sequence and gene structure, including the exon 6 region harboring the founder mutation. Herein, we describe a new mouse model of this form of limb-girdle muscular dystrophy generated using CRISPR/Cas9-mediated gene editing to introduce a single thymine deletion in murine exon 6, recreating the 521ΔT point mutation in Sgcg These mice express the 521ΔT transcript, lack γ-sarcoglycan protein and exhibit a severe dystrophic phenotype. Phenotypic characterization demonstrated reduced muscle mass, increased sarcolemmal leak and fragility, and decreased muscle function, consistent with the human pathological findings. Furthermore, we showed that intramuscular administration of a murine-specific multiple exon-directed antisense oligonucleotide cocktail effectively corrected the 521ΔT reading frame. These data demonstrate a molecularly and pathologically suitable model for in vivo testing of a multi-exon skipping strategy to advance preclinical development of this genetic correction approach.

Deletion of Sulfonylurea Receptor 2 in the Adult Myocardium Enhances Cardiac Glucose Uptake and Is Cardioprotective.

The adult myocardium relies on oxidative metabolism. In ischemic myocardium, such as the embryonic heart, glycolysis contributes more prominently as a fuel source. The sulfonylurea receptor 2 (SUR2) was previously implicated in the normal myocardial transition from glycolytic to oxidative metabolism that occurs during adaptation to postnatal life. This receptor was now selectively deleted in adult mouse myocardium resulting in protection from ischemia reperfusion injury. SUR2-deleted cardiomyocytes had enhanced glucose uptake, and SUR2 forms a complex with the major glucose transporter. These data identify the SUR2 receptor as a target to shift cardiac metabolism to protect against myocardial injury.

Dusp6 is a genetic modifier of growth through enhanced ERK activity.

Like other single-gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole-genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the dual-specificity phosphatase 6 (Dusp6) gene from the DBA/2 J (D2) mouse strain. DUSP6 is a broadly expressed dual-specificity phosphatase protein, which binds and dephosphorylates extracellular-signal-regulated kinase (ERK), leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DUSP6 and ERK resulting in increased ERK phosphorylation and ERK activity. In dystrophic muscle, DUSP6 Met62Ile is strongly upregulated to counteract its reduced activity. We found that myoblasts from the D2 background were insensitive to a specific small molecule inhibitor of DUSP6, while myoblasts expressing the canonical DUSP6 displayed enhanced proliferation after exposure to DUSP6 inhibition. These data identify DUSP6 as an important regulator of ERK activity in the setting of muscle growth and muscular dystrophy.

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor ß (TGFß) pathway, osteopontin encoded by the SPP1 gene and latent TGFß binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFß and TGFß-associated pathways. We identified that increased TGFß resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFß and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.

Experimental Modeling Supports a Role for MyBP-HL as a Novel Myofilament Component in Arrhythmia and Dilated Cardiomyopathy.

BACKGROUND: Cardiomyopathy and arrhythmias are under significant genetic influence. Here, we studied a family with dilated cardiomyopathy and associated conduction system disease in whom prior clinical cardiac gene panel testing was unrevealing. METHODS: Whole-genome sequencing and induced pluripotent stem cells were used to examine a family with dilated cardiomyopathy and atrial and ventricular arrhythmias. We also characterized a mouse model with heterozygous and homozygous deletion of Mybphl. RESULTS: Whole-genome sequencing identified a premature stop codon, R255X, in the MYBPHL gene encoding MyBP-HL (myosin-binding protein-H like), a novel member of the myosin-binding protein family. MYBPHL was found to have high atrial expression with low ventricular expression. We determined that MyBP-HL protein was myofilament associated in the atria, and truncated MyBP-HL protein failed to incorporate into the myofilament. Human cell modeling demonstrated reduced expression from the mutant MYBPHL allele. Echocardiography of Mybphl heterozygous and null mouse hearts exhibited a 36% reduction in fractional shortening and an increased diastolic ventricular chamber size. Atria weight normalized to total heart weight was significantly increased in Mybphl heterozygous and null mice. Using a reporter system, we detected robust expression of Mybphl in the atria, and in discrete puncta throughout the right ventricular wall and septum, as well. Telemetric electrocardiogram recordings in Mybphl mice revealed cardiac conduction system abnormalities with aberrant atrioventricular conduction and an increased rate of arrhythmia in heterozygous and null mice. CONCLUSIONS: The findings of reduced ventricular function and conduction system defects in Mybphl mice support that MYBPHL truncations may increase risk for human arrhythmias and cardiomyopathy.

Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy.

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

Overexpression of Latent TGFß Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFß.

Latent TGFß binding proteins (LTBPs) regulate the extracellular availability of latent TGFß. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFß family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFß and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFß family ligand binding protein with the capacity to modify muscle disease through overexpression.

Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping.

Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations.

Targeting latent TGFß release in muscular dystrophy.

Latent transforming growth factor-ß (TGFß) binding proteins (LTBPs) bind to inactive TGFß in the extracellular matrix. In mice, muscular dystrophy symptoms are intensified by a genetic polymorphism that changes the hinge region of LTBP, leading to increased proteolytic susceptibility and TGFß release. We have found that the hinge region of human LTBP4 was also readily proteolysed and that proteolysis could be blocked by an antibody to the hinge region. Transgenic mice were generated to carry a bacterial artificial chromosome encoding the human LTBP4 gene. These transgenic mice displayed larger myofibers, increased damage after muscle injury, and enhanced TGFß signaling. In the mdx mouse model of Duchenne muscular dystrophy, the human LTBP4 transgene exacerbated muscular dystrophy symptoms and resulted in weaker muscles with an increased inflammatory infiltrate and greater LTBP4 cleavage in vivo. Blocking LTBP4 cleavage may be a therapeutic strategy to reduce TGFß release and activity and decrease inflammation and muscle damage in muscular dystrophy.

Abcc9 is required for the transition to oxidative metabolism in the newborn heart.

The newborn heart adapts to postnatal life by shifting from a fetal glycolytic metabolism to a mitochondrial oxidative metabolism. Abcc9, an ATP-binding cassette family member, increases expression concomitant with this metabolic shift. Abcc9 encodes a membrane-associated receptor that partners with a potassium channel to become the major potassium-sensitive ATP channel in the heart. Abcc9 also encodes a smaller protein enriched in the mitochondria. We now deleted exon 5 of Abcc9 to ablate expression of both plasma membrane and mitochondria-associated Abcc9-encoded proteins, and found that the myocardium failed to acquire normal mature metabolism, resulting in neonatal cardiomyopathy. Unlike wild-type neonatal cardiomyocytes, mitochondria from Ex5 cardiomyocytes were unresponsive to the KATP agonist diazoxide, consistent with loss of KATP activity. When exposed to hydrogen peroxide to induce cell stress, Ex5 neonatal cardiomyocytes displayed a rapid collapse of mitochondria membrane potential, distinct from wild-type cardiomyocytes. Ex5 cardiomyocytes had reduced fatty acid oxidation, reduced oxygen consumption and reserve. Morphologically, Ex5 cardiac mitochondria exhibited an immature pattern with reduced cross-sectional area and intermitochondrial contacts. In the absence of Abcc9, the newborn heart fails to transition normally from fetal to mature myocardial metabolism.-Fahrenbach, J. P., Stoller, D., Kim, G., Aggarwal, N., Yerokun, B., Earley, J. U., Hadhazy, M., Shi, N.-Q., Makielski, J. C., McNally, E. M. Abcc9 is required for the transition to oxidative metabolism in the newborn heart.

Dysferlin and myoferlin regulate transverse tubule formation and glycerol sensitivity.

Dysferlin is a membrane-associated protein implicated in muscular dystrophy and vesicle movement and function in muscles. The precise role of dysferlin has been debated, partly because of the mild phenotype in dysferlin-null mice (Dysf). We bred Dysf mice to mice lacking myoferlin (MKO) to generate mice lacking both myoferlin and dysferlin (FER). FER animals displayed progressive muscle damage with myofiber necrosis, internalized nuclei, and, at older ages, chronic remodeling and increasing creatine kinase levels. These changes were most prominent in proximal limb and trunk muscles and were more severe than in Dysf mice. Consistently, FER animals had reduced ad libitum activity. Ultrastructural studies uncovered progressive dilation of the sarcoplasmic reticulum and ectopic and misaligned transverse tubules in FER skeletal muscle. FER muscle, and Dysf- and MKO-null muscle, exuded lipid, and serum glycerol levels were elevated in FER and Dysf mice. Glycerol injection into muscle is known to induce myopathy, and glycerol exposure promotes detachment of transverse tubules from the sarcoplasmic reticulum. Dysf, MKO, and FER muscles were highly susceptible to glycerol exposure in vitro, demonstrating a dysfunctional sarcotubule system, and in vivo glycerol exposure induced severe muscular dystrophy, especially in FER muscle. Together, these findings demonstrate the importance of dysferlin and myoferlin for transverse tubule function and in the genesis of muscular dystrophy.

The superhealing MRL background improves muscular dystrophy.

BACKGROUND: Mice from the MRL or "superhealing" strain have enhanced repair after acute injury to the skin, cornea, and heart. We now tested an admixture of the MRL genome and found that it altered the course of muscle pathology and cardiac function in a chronic disease model of skeletal and cardiac muscle. Mice lacking γ-sarcoglycan (Sgcg), a dystrophin-associated protein, develop muscular dystrophy and cardiomyopathy similar to their human counterparts with limb girdle muscular dystrophy. With disruption of the dystrophin complex, the muscle plasma membrane becomes leaky and muscles develop increased fibrosis. METHODS: MRL/MpJ mice were bred with Sgcg mice, and cardiac function was measured. Muscles were assessed for fibrosis and membrane leak using measurements of hydroxyproline and Evans blue dye. Quantitative trait locus mapping was conducted using single nucleotide polymorphisms distinct between the two parental strains. RESULTS: Introduction of the MRL genome reduced fibrosis but did not alter membrane leak in skeletal muscle of the Sgcg model. The MRL genome was also associated with improved cardiac function with reversal of depressed fractional shortening and the left ventricular ejection fraction. We conducted a genome-wide analysis of genetic modifiers and found that a region on chromosome 2 was associated with cardiac, diaphragm muscle and abdominal muscle fibrosis. CONCLUSIONS: These data are consistent with a model where the MRL genome acts in a dominant manner to suppress fibrosis in this chronic disease setting of heart and muscle disease.

Cardiomyocyte sulfonylurea receptor 2-KATP channel mediates cardioprotection and ST segment elevation.

Sulfonylurea receptor-containing ATP-sensitive potassium (K(ATP)) channels have been implicated in cardioprotection, but the cell type and constitution of channels responsible for this protection have not been clear. Mice deleted for the first nucleotide binding region of sulfonylurea receptor 2 (SUR2) are referred to as SUR2 null since they lack full-length SUR2 and glibenclamide-responsive K(ATP) channels in cardiac, skeletal, and smooth muscle. As previously reported, SUR2 null mice develop electrocardiographic changes of ST segment elevation that were shown to correlate with coronary artery vasospasm. Here we restored expression of the cardiomyocyte SUR2-K(ATP) channel in SUR2 null mice by generating transgenic mice with ventricular cardiomyocyte-restricted expression of SUR2A. Introduction of the cardiomyocyte SUR2A transgene into the SUR2 null background restored functional cardiac K(ATP) channels. Hearts isolated from rescued mice, referred to as MLC2A, had significantly reduced infarct size (27 ± 3% of area at risk) compared with SUR2 null mice (36 ± 3% of area at risk). Compared with SUR2 null hearts, MLC2A hearts exhibited significantly improved cardiac function during the postischemia reperfusion period primarily because of preservation of low diastolic pressures. Additionally, restoration of cardiac SUR2-K(ATP) channels significantly reduced the degree and frequency of ST segment elevation episodes in MLC2A mice. Therefore, cardioprotective mechanisms both dependent and independent of SUR2-K(ATP) channels contribute to cardiac function.

Latent TGF-beta-binding protein 4 modifies muscular dystrophy in mice.

Most single-gene diseases, including muscular dystrophy, display a nonuniform phenotype. Phenotypic variability arises, in part, due to the presence of genetic modifiers that enhance or suppress the disease process. We employed an unbiased mapping approach to search for genes that modify muscular dystrophy in mice. In a genome-wide scan, we identified a single strong locus on chromosome 7 that influenced two pathological features of muscular dystrophy, muscle membrane permeability and muscle fibrosis. Within this genomic interval, an insertion/deletion polymorphism of 36 bp in the coding region of the latent TGF-beta-binding protein 4 gene (Ltbp4) was found. Ltbp4 encodes a latent TGF-beta-binding protein that sequesters TGF-beta and regulates its availability for binding to the TGF-beta receptor. Insertion of 12 amino acids into the proline-rich region of LTBP4 reduced proteolytic cleavage and was associated with reduced TGF-beta signaling, decreased fibrosis, and improved muscle pathology in a mouse model of muscular dystrophy. In contrast, a 12-amino-acid deletion in LTBP4 was associated with increased proteolysis, SMAD signaling, and fibrosis. These data identify Ltbp4 as a target gene to regulate TGF-beta signaling and modify outcomes in muscular dystrophy.